Microfluidic matrix localization apparatus and methods

ABSTRACT

Multiphasic microfluidic apparatus for performing product fluid manipulation and separation in a single continuous unit are provided. Related methods, kits, and compositions are also provided.

BACKGROUND OF THE INVENTION

[0001] Manipulating fluidic reagents and assessing the results ofreagent interactions are central to chemical and biological science.Manipulations include mixing fluidic reagents, assaying productsresulting from such mixtures, and separation or purification of productsor reagents and the like. Assessing the results of reagent interactionscan include autoradiography, spectroscopy, microscopy, photography, massspectrometry, nuclear magnetic resonance and many other techniques forobserving and recording the results of mixing reagents. A singleexperiment may involve literally hundreds of fluidic manipulations,product separations, result recording processes and data compilation andintegration steps. Fluidic manipulations are performed using a widevariety of laboratory equipment, including various fluid heatingdevices, fluidic mixing devices, centrifugation equipment, moleculepurification apparatus, chromatographic machinery, gel electrophoreticequipment and the like. The effects of mixing fluidic reagents aretypically assessed by additional equipment relating to detection,visualization or recording of an event to be assayed, such asspectrophotometers, autoradiographic equipment, microscopes, gelscanners, computers and the like.

[0002] Because analysis of even simple chemical, biochemical, orbiological phenomena requires many different types of laboratoryequipment, the modern laboratory is complex, large and expensive. Inaddition, because so many different types of equipment are used in evenconceptually simple experiments such as DNA synthesis or sequencing, ithas not generally been practical to integrate different types ofequipment to improve automation. The need for a laboratory worker tophysically perform many aspects of laboratory science imposes sharplimits on the number of experiments which a laboratory can perform, andincreases the undesirable exposure of laboratory workers to toxic orradioactive reagents.

[0003] One particularly labor intensive biochemical series of laboratoryfluidic manipulations is nucleic acid synthesis and analysis. A varietyof in vitro amplification methods for biochemical synthesis of nucleicacids are available, such as the polymerase chain reaction (PCR). See,Mullis et al., (1987) U.S. Pat. No. 4,683,202 and PCR Protocols A Guideto Methods and Applications (Innis et al. eds, Academic Press Inc. SanDiego, Calif. (1990) (Innis). PCR methods typically require the use ofspecialized machinery for performing thermocycling reactions to performDNA synthesis, followed by the use of specialized machinery forelectrophoretic analysis of synthesized DNA. For a description ofnucleic acid manipulation methods and apparatus see Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1997, supplement 37) (Ausubel).

[0004] Another particularly important and labor intensive biochemicalseries of laboratory fluidic manipulations which are typically performedon nucleic acids which are made recombinantly or synthetically isnucleic acid sequencing. Efficient DNA sequencing technology is centralto the development of the biotechnology industry and basic biologicalresearch. Improvements in the efficiency and speed of DNA sequencing areneeded to keep pace with the demands for DNA sequence information. TheHuman Genome Project, for example, has set a goal of dramaticallyincreasing the efficiency, cost-effectiveness and throughput of DNAsequencing techniques. See, e.g., Collins, and Galas (1993) Science262:43-46.

[0005] Most DNA sequencing today is carried out by chain terminationmethods of DNA sequencing. The most popular chain termination methods ofDNA sequencing are variants of the dideoxynucleotide mediated chaintermination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad.Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing,see, Ausubel or Sambrook, supra. Four color sequencing is described inU.S. Pat. No. 5,171,534. Thousands of laboratories employdideoxynucleotide chain termination techniques. Commercial kitscontaining the reagents most typically used for these methods of DNAsequencing are available and widely used.

[0006] In addition to the Sanger methods of chain termination, new PCRexonuclease digestion methods have also been proposed for DNAsequencing. Direct sequencing of PCR generated amplicons by selectivelyincorporating boronated nuclease resistant nucleotides into theamplicons during PCR and digestion of the amplicons with a nuclease toproduce sized template fragments has been proposed (Porter et al. (1997)Nucleic Acids Research 25(8):1611-1617). In the methods, 4 PCR reactionson a template are performed, in each of which one of the nucleotidetriphosphates in the PCR reaction mixture is partially substituted witha 2′deoxynucleoside 5′-α[P-borano]-triphosphate. The boronatednucleotide is stocastically incorporated into PCR products at varyingpositions along the PCR amplicon in a nested set of PCR fragments of thetemplate. An exonuclease which is blocked by incorporated boronatednucleotides is used to cleave the PCR amplicons. The cleaved ampliconsare then separated by size using polyacrylamide gel electrophoresis,providing the sequence of the amplicon. An advantage of this method isthat it requires fewer biochemical manipulations than performingstandard Sanger-style sequencing of PCR amplicons.

[0007] Other sequencing methods which reduce the number of stepsnecessary for template preparation and primer selection have beendeveloped. One proposed variation on sequencing technology involves theuse of modular primers for use in PCR and DNA sequencing. For example,Ulanovsky and co-workers have described the mechanism of the modularprimer effect (Beskin et al. (1995) Nucleic Acids Research23(15):2881-2885) in which short primers of 5-6 nucleotides canspecifically prime a template-dependent polymerase enzyme for templatedependent nucleic acid synthesis. A modified version of the use of themodular primer strategy, in which small nucleotide primers arespecifically elongated for use in PCR to amplify and sequence templatenucleic acids has also been described. The procedure is referred to asDNA sequencing using differential extension with nucleotide subsets(DENS). See, Raja et al. (1997) Nucleic Acids Research 25(4):800-805.

[0008] Improvements in methods for generating sequencing templates havealso been developed. DNA sequencing typically involves three steps: i)making suitable templates for the regions to be sequenced (i.e., bysynthesizing or cloning the nucleic acid to be sequenced); ii) runningsequencing reactions for electrophoresis, and iii) assessing the resultsof the reaction. The latter steps are sometimes automated by use oflarge and very expensive workstations and autosequencers. The first stepoften requires careful experimental design and laborious DNAmanipulation such as the construction of nested deletion mutants. See,Griffin, H. G. and Griffin, A. M. (1993) DNA sequencing protocols,Humana Press, New Jersey. Alternatively, random “shot-gun” sequencingmethods, are sometimes used to make templates, in which randomlyselected sub clones, which may or may not have overlapping sequenceinformation, are randomly sequenced. The sequences of the sub clones arecompiled to produce an ordered sequence. This procedure eliminatescomplicated DNA manipulations; however, the method is inherentlyinefficient because many recombinant clones must be sequenced due to therandom nature of the procedure. Because of the labor intensive nature ofsequencing, the repetitive sequencing of many individual clonesdramatically reduces the throughput of these sequencing systems.

[0009] Recently, Hagiwara and Curtis (1996) Nucleic Acids Research24(12):2460-2461 developed a “long distance sequencer” PCR protocol forgenerating overlapping nucleic acids from very large clones tofacilitate sequencing, and methods of amplifying and tagging theoverlapping nucleic acids into suitable sequencing templates. Themethods can be used in conjunction with shotgun sequencing techniques toimprove the efficiency of shotgun methods.

[0010] In an attempt to increase laboratory throughput and to decreaseexposure of laboratory workers to reagents, various strategies have beenperformed. For example, robotic introduction of fluids onto microtiterplates is commonly performed to speed mixing of reagents and to enhanceexperimental throughput. More recently, microscale devices for highthroughput mixing and assaying of small fluid volumes have beendeveloped. For example, U.S. Ser. No. 08/761,575 entitled “HighThroughput Screening Assay Systems in Microscale Fluidic Devices” byParce et al. provides pioneering technology related to microscalefluidic devices, especially including electrokinetic devices. Thedevices are generally suitable for assays relating to the interaction ofbiological and chemical species, including enzymes and substrates,ligands and ligand binders, receptors and ligands, antibodies andantibody ligands, as well as many other assays. Because the devicesprovide the ability to mix fluidic reagents and assay mixing results ina single continuous process, and because minute amounts of reagents canbe assayed, these microscale devices represent a fundamental advance forlaboratory science. Pioneering integrated systems for nucleic acidsequencing utilizing microfluidic fluid manipulation are described in,e.g., provisional patent application U.S. S No. 60/068,311, entitled“Closed Loop Biochemical Analyzer” by Knapp, filed Dec. 19, 1997 and“Closed Loop Biochemical Analyzers” by Knapp et al. U.S. Ser. No. ______(attorney docket number 017646-0003100US) filed Apr. 3, 1998.

[0011] In the electrokinetic microscale devices and systems provided byParce et al. and Knapp above, an appropriate fluid is flowed into amicrochannel etched in a substrate having functional groups present atthe surface. The groups ionize when the surface is contacted with anaqueous solution. For example, where the surface of the channel includeshydroxyl functional groups at the surface, e.g., as in glass substrates,protons can leave the surface of the channel and enter the fluid. Undersuch conditions, the surface possesses a net negative charge, whereasthe fluid will possess an excess of protons, or positive charge,particularly localized near the interface between the channel surfaceand the fluid. By applying an electric field along the length of thechannel, cations will flow toward the negative electrode. Movement ofthe sheath of positively charged species in the fluid pulls the solventwith them.

[0012] Although improvements in robotic manipulation of fluidic reagentsand miniaturization of laboratory equipment have been made, and althoughparticular biochemical processes such as DNA amplification andsequencing are very well developed, there still exists a need foradditional techniques and apparatus for mixing and assaying fluidicreagents, for integration of such systems and for reduction of thenumber of manipulations required to perform biochemical manipulationssuch as DNA sequencing. Ideally, these new apparatus would be usefulwith, and compatible to, established biochemical protocols. Thisinvention provides these and many other features.

SUMMARY OF THE INVENTION

[0013] The present invention provides multi-phasic microfluidicapparatus which are suitable for performing fluidic mixing followed byseparation of mixing products. These apparatus have fluid mixing regionssuch as microfluidic channels and fluidly connected product separationregions, e.g., microchannels comprising a sieving matrix such as aseparations gel. Thus, mixing reactions, including, e.g., PCR, can beperformed in a first fluidic phase, followed by a separation reaction ina second fluidic separations phase. The fluid connection regions aretypically unvalved, and/or electrically gated and optionally compriseelectrical control elements.

[0014] Several techniques for making multi-phasic microfluidic apparatusare also provided. In one embodiment, a first phase such as a gel, isselectively cross-linked at regions of a microchannel where gel isdesired (e.g. by photopolymerizing the gel in place). In otherembodiments, pressure (negative or positive) is used to force onefluidic phase into a specified portion of a microchannel. Differentfluidic phases are optionally loaded electrokinetically. Othertechniques are also shown.

[0015] In operation, components are typically made or purified in afirst phase (e.g., a PCR amplification mixture, DNA sequencing reaction,binding reaction, enzyme reaction, or the like) and separated in asecond phase such as a sieving matrix. In a preferred embodiment,products produced in the first phase are electrically loaded on thesecond phase, where the components initially stack and then separate asthey electrophorese through the second phase.

[0016] In addition, it is surprisingly discovered that the PCR reactioncan be performed in the presence of a variety of sieving matrices,including: agarose, linear polyacrylamide, methylcellulose, polyethyleneoxide and hydroxy ethyl cellulose and that resulting PCR products areseparable in the microfluidic devices herein. Accordingly, elegant chipdesigns for performing PCR in microfluidic chips are shown for mixingPCR reaction components and sieving matrixes, performing thermocyclingreactions and separating resulting PCR products in microfluidicchannels.

BRIEF SUMMARY OF THE FIGURES

[0017]FIG. 1 is a schematic drawing of the “12 A” chip.

[0018]FIG. 2 is a schematic drawing of a microchannel for joule heating.

[0019]FIG. 3 is a schematic drawing of the 12 A chip and a graph of PCRproduct separation.

[0020]FIG. 4 shows two graphs of data from PCR of CFTR from whole blood.

[0021]FIG. 5 is a graph of data from PCR amplification of purified WBCs.

[0022]FIG. 6 is a graph of data from PCR amplification ofelectrophoresed WBCs.

[0023]FIG. 7 is a graph of data from PCR amplification ofelectrophoresed WBCs and PCR amplification of purified WBCs.

[0024]FIG. 8 is a graph of HLA PCR with 0.5% methylcellulose.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Many laboratory fluidic operations are highly cumbersome,requiring many steps and a high labor input. This high labor input makesthe efficiency of the modern laboratory relatively low compared to manymanufacturing or service industries. Accordingly, extensive attempts toimprove efficiency for laboratory fluidic operations have been made,utilizing robotics and miniaturization. Most recently, microfluidicoperations using electrokinetic microfluidic apparatus haverevolutionized laboratory operations. See, e.g., International PatentApplication No. WO 96/04547 to Ramsey et al., as well as U.S. Ser. No.08/761,575 by Parce et al. U.S. Ser. No. 08/845,754 to Dubrow et al.U.S. S No. 60/068311, by Knapp and U.S. Ser. No. 08/835,101 by Knapp etal.

[0026] The invention provides apparatus and related kits, methods ofmaking the apparatus and kits and methods of using the apparatus andkits. These apparatus and related methods provide multi-phasicmicrofluidic apparatus suitable for performing integrated fluidicoperations. Using the apparatus and methods, it is possible to performmany or all of the fluidic operations needed for an experiment ordiagnostic procedure in an integrated fashion in a single apparatus. Thepresent invention provides apparatus, systems and methods fordramatically increasing the speed and simplicity of screening,manipulating and assessing fluidic reagents, reagent mixtures, reactionproducts (including the products of DNA sequencing reactions) and thelike. The invention provides integrated systems for performing a varietyof chemical, biochemical and biological experiments and other fluidicoperations, including PCR, DNA sequencing, integrated or sequentialscreening of chemical or biological libraries, and the like. Althoughthe microfluidic systems of the invention are generally described interms of the performance of chemical, biochemical or biologicalreactions separations, incubations and the like, it will be understoodthat, as fluidic systems having general applicability, these systems canhave a wide variety of different uses, e.g., as metering or dispensingsystems in both biological and nonbiological applications.

Making Multi-Phasic Microfluidic Apparatus

[0027] The apparatus of the invention are typically “multi-phasic,”although uni-phasic embodiments, particularly for PCR amplification, arealso contemplated. The phrase “multi-phasic” is intended to indicate amicrofluidic apparatus which includes microchannels, microchambers,microwells or the like, where there are at least two different fluidicphases present in the apparatus. For example, in one embodiment,crossing microchannels comprise different fluidic phases. For example, afirst fluidic phase (e.g., a biological or chemical reaction mixture) ina first microchannel can cross a second microchannel comprising a secondfluidic phase (e.g., a sieving matrix) adapted to analyzing componentsof the first fluidic phase.

[0028] An example simple apparatus of the invention is provided byFIG. 1. Fluidic reaction channel 101 and fluidic separations channel 103are filled with separation matrix 105 which can be any separationsmatrix as described more fully below, including a polyacrylamide gel orsolution. Separation matrix 105 is replaced in fluidic channel 101,e.g., by flowing buffer 107 into separation channel 101 under pressure,thereby forcing separation matrix 105 out of channel 101. A variety ofvariations on this apparatus are discussed herein.

[0029] Making Microfluidic Substrates

[0030] The microfluidic devices of the invention typically comprise asubstrate or body having microfluidic channels, chambers, wells or thelike disposed therein, e.g., as depicted in FIG. 1. In the apparatus ofthe invention, at least two different fluid phases are also disposedwithin the substrate, e.g., in a plurality of reaction channels.

[0031] Manufacturing of microscale elements into the surface ofsubstrates is generally carried out by any number of microfabricationtechniques that are known in the art. For example, lithographictechniques are employed in fabricating, e.g., glass, quartz or siliconsubstrates, using methods well known in the semiconductor manufacturingindustries such as photolithographic etching, plasma etching or wetchemical etching. See, Sorab K. Ghandi, VLSI Principles: Silicon andGallium Arsenide, NY, Wiley (see, esp. Chapter 10). Alternatively,micromachining methods such as laser drilling, air abrasion,micromilling and the like are employed. Similarly, for polymericsubstrates, well known manufacturing techniques are used. Thesetechniques include injection molding or stamp molding methods wherelarge numbers of substrates are produced using, e.g., rolling stamps toproduce large sheets of microscale substrates or polymer microcastingtechniques where the substrate is polymerized within a micromachinedmold. Polymeric substrates are further described in Provisional PatentApplication Serial No. 60/015,498, filed Apr. 16, 1996 (Attorney DocketNo. 017646-002600), and U.S. Ser. No. 08/843,212, filed Apr. 14, 1997.

[0032] In addition to micromachining methods, printing methods are alsoused to fabricate chambers channels and other microfluidic elements on asolid substrate. Such methods are taught in detail in U.S. Ser. No.08/987,803 by Colin Kennedy, Attorney Docket Number 017646-004400, filedDec. 10, 1997 entitled “Fabrication of Microfluidic Circuits by PrintingTechniques.” In brief, printing methods such as ink-jet printing, laserprinting or other printing methods are used to print the outlines of amicrofluidic element on a substrate, and a cover layer is fixed over theprinted outline to provide a closed microfluidic element.

[0033] The substrates of the invention optionally include a planarelement which overlays the channeled portion of the substrate, enclosingand fluidly sealing the various channels, wells and other microfluidicelements. Attaching the planar cover element is achieved by a variety ofmeans, including, e.g., thermal bonding, adhesives or, in the case ofcertain substrates, e.g., glass, or semi-rigid and non-rigid polymericsubstrates, a natural adhesion between the two components. The planarcover element can additionally be provided with access ports and/orreservoirs for introducing the various fluid elements needed for aparticular screen, and for introducing electrodes for electrokineticmovement.

[0034] Typically, an individual microfluidic device will have an overallsize that is fairly small. Generally, the devices will have a square orrectangular shape, but the specific shape of the device can be easilyvaried to accommodate the users needs. While the size of the device isgenerally dictated by the number of operations performed within a singledevice, such devices will typically be from about 1 to about 20 cmacross, and from about 0.01 to about 1.0 cm thick. In a preferredaspect, the devices include glass substrates with channels or othermicrofluidic elements fabricated therein or thereon. These substratesare typically easy to produce, making them potentially disposable.

[0035] Although microfabricated fluid pumping and valving systems arereadily employed in the devices of the invention, the cost andcomplexity associated with their manufacture and operation can generallyprohibit their use in mass-produced and potentially disposable devicesas are envisioned by the present invention. The devices of the inventionwill typically include an electroosmotic fluid direction system. Suchfluid direction systems combine the elegance of a fluid direction systemdevoid of moving parts, with an ease of manufacturing, fluid control anddisposability. Examples of particularly preferred electroosmotic fluiddirection systems include, e.g., those described in International PatentApplication No. WO 96/04547 to Ramsey et al., as well as U.S. Ser. No.08/761,575 by Parce et al. and U.S. Ser. No. 08/845,754 by Dubrow et al.

[0036] In brief, these fluidic control systems typically includeelectrodes disposed within reservoirs that are placed in fluidconnection with the channels fabricated into the surface of thesubstrate. The materials stored in the reservoirs are transportedthrough the channel system delivering appropriate volumes of the variousmaterials to one or more regions on the substrate in order to carry outa desired screening assay.

[0037] Material transport and direction is accomplished throughelectrokinesis, e.g., electroosmosis or electrophoresis. As used herein,“electrokinetic material transport systems” or “electrokinetic devices”include systems which transport and direct materials within aninterconnected channel and/or chamber containing structure, through theapplication of electrical fields to the materials, thereby causingmaterial movement through and among the channel and/or chambers, i.e.,cations will move toward the negative electrode, while anions will movetoward the positive electrode. Such electrokinetic material transportand direction systems include those systems that rely upon theelectrophoretic mobility of charged species within the electric fieldapplied to the structure. Such systems are more particularly referred toas electrophoretic material transport systems. Other electrokineticmaterial direction and transport systems rely upon the electroosmoticflow of fluid and material within a channel or chamber structure whichresults from the application of an electric field across suchstructures.

[0038] In brief, when an appropriate fluid is placed in a channel orother fluid conduit having functional groups present at the surface,those groups can ionize. For example, where the surface of the channelincludes hydroxyl functional groups at the surface, protons can leavethe surface of the channel and enter the fluid. Under such conditions,the surface will possess a net negative charge, whereas the fluid willpossess an excess of protons or positive charge, particularly localizednear the interface between the channel surface and the fluid. Byapplying an electric field along the length of the channel, cations willflow toward the negative electrode. Movement of the positively chargedspecies in the fluid pulls the solvent with them. An electrokineticdevice moves components by applying an electric field to the components,typically in a microfluidic channel. By applying an electric field alongthe length of the channel, cations will flow toward a negativeelectrode, while anions will flow towards a positive electrode. Movementof charged species in the fluid pulls the solvent with the fluid. Thesteady state velocity of this fluid movement is generally given by theequation: $v = \frac{{\varepsilon\xi}\quad E}{4\pi \quad \eta}$

[0039] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. The solvent velocity is,therefore, directly proportional to the surface potential.

[0040] To provide appropriate electric fields, the system generallyincludes a voltage controller that is capable of applying selectablevoltage levels, simultaneously, to each of the reservoirs, includingground. Such a voltage controller can be implemented using multiplevoltage dividers and multiple relays to obtain the selectable voltagelevels. Alternatively, multiple, independent voltage sources are used.The voltage controller is electrically connected to each of thereservoirs via an electrode positioned or fabricated within each of theplurality of reservoirs. In one embodiment, multiple electrodes arepositioned to provide for switching of the electric field direction in amicrochannel, thereby causing the analytes to travel a longer distancethan the physical length of the microchannel. Use of electrokinetictransport to control material movement in interconnected channelstructures was described in WO 96/04547 to Ramsey, which is incorporatedby reference.

[0041] Substrate materials are also selected to produce channels havinga desired surface charge. In the case of glass substrates, the etchedchannels will possess a net negative charge resulting from the ionizedhydroxyls naturally present at the surface. Alternatively, surfacemodifications are employed to provide an appropriate surface charge,e.g., coatings, derivatization, e.g., silanation, or impregnation of thesurface to provide appropriately charged groups on the surface. Examplesof such treatments are described in, e.g., Provisional PatentApplication Serial No. 60/015,498, filed Apr. 16, 1996 (Attorney DocketNo. 017646-002600). See also, Attorney Docket Number 17646-002610, filedApr. 14, 1997.

[0042] Modulating voltages are then concomitantly applied to the variousreservoirs to affect a desired fluid flow characteristic, e.g.,continuous or discontinuous (e.g., a regularly pulsed field causing theflow to oscillate direction of travel) flow of receptor/enzyme,ligand/substrate toward the waste reservoir with the periodicintroduction of test compounds. Particularly, modulation of the voltagesapplied at the various reservoirs can move and direct fluid flow throughthe interconnected channel structure of the device in a controlledmanner to effect the fluid flow for the desired screening assay andapparatus.

[0043] While a number of devices for carrying out particular methodsaccording to the invention are described in substantial detail herein,it will be recognized that the specific configuration of these deviceswill generally vary depending upon the type of manipulation or reactionto be performed. The small scale, integratability and self-containednature of these devices allows for virtually any reaction orientation tobe realized within the context of the microlaboratory system.

[0044] Because the microfluidic devices of the invention preferablyemploy electroosmotic fluid direction systems, and are substantiallysealed to the outside environment, excepting reagent, buffer or sampleports, they are capable of performing fluidic operations whilemaintaining precise control of the amounts of different fluids to bedelivered to the different regions of the substrate.

[0045] Adding Multiple Phases to Microfluidic Substrates

[0046] One aspect of the invention is the placement of different fluidicphases in different regions of a microfluidic substrate. For example, itis advantageous to provide a microfluidic substrate with reactioncomponents in one region and sieving matrix in other portions for theseparation of reaction components. Accordingly, one aspect of theinvention is the selective placement of fluidic phases in selectedchannels or channel regions of a microfluidic substrate. These materials(or precursors of the materials, e.g., monomers to be polymerized insubsequent steps as discussed below) are loaded into microfluidiccomponents by electrokinesis or by pressurized pumping.

[0047] A wide variety of sieving and molecular partition matrixes areavailable, and can be used in the multi-phasic apparatus of theinvention. For example, a variety of sieving matrixes, partitionmatrixes and the like are available from Supelco, Inc. (Bellefonte, Pa.;see, 1997 Suppleco catalogue). Common matrixes which are useful in thepresent invention include those generally used in low pressure liquidchromatography, gel electrophoresis and other liquid phase separations;matrix materials designed primarily for non-liquid phase chromatographyare also useful in certain contexts, as the materials often retainseparatory characteristics when suspended in fluids. For a discussion ofelectrophoresis see, e.g., Weiss (1995) Ion Chromatography VCHPublishers Inc.; Baker (1995) Capillary Electrophoresis John Wiley andSons; Kuhn (1993) Capillary Electrophoresis: Principles and PracticeSpringer Verlag; Righetti (1996) Capillary Electrophoresis in AnalyticalBiotechnology CRC Press; Hill (1992) Detectors for CapillaryChromatography John Wiley and Sons; Gel Filtration: Principles andMethods (5th Edition) Pharmacia; Gooding and Regnier (1990) HPLC ofBiological Macromolecules: Methods and Applications (Chrom. Sci. Series,volume 51) Marcel Dekker and Scott (1995) Techniques and Practices ofChromatography Marcel Dekker, Inc.

[0048] Commercially available low pressure liquid chromatography mediainclude, e.g., non-ionic macroreticular and macroporous resins whichadsorb and release components based upon hydrophilic or hydrophobicinteractions such as Amberchrom resins (highly cross-linkedstyrene/divinylbenzene copolymers suitable for separation of peptides,proteins, nucleic acids, antibiotics, phytopharmacologicals, andvitamins); the related Amberlite XAD series resins (polyaromatics andacrylic esters) and amberchroms (polyaromatic and polymethacrylates)(manufactured by Rohm and Haas, available through Suppleco); Diaion(polyaromatic or polymethacrylic beads); Dowex (polyaromatics orsubstituted hydrophilic functionalized polyaromatics) (manufactured byDow Chemical, available through Suppleco); Duolite (phenol-formaldehydewith methanolic functionality), MCI GEL sephabeads, supelite DAX-8(acrylic ester) and Supplepak (polyaromatic) (all of the precedingmaterials are available from Suppleco). For a description of uses forAmberlite and Duolite resins, see, Amberlite/Duolite Anion ExchangeResins (Avaliable from Suppleco, Cat No. T412141). Gel filtrationchromatography matrixes are also suitable, including sephacryl,sephadex, sepharose, superdex, superose, toyopearl, agarose, cellulose,dextrans, mixed bead resins, polystyrene, nuclear resins, DEAEcellulose, Benzyl DEA cellulose, TEAE cellulose, and the like(Suppleco).

[0049] Gel electrophoresis media include silica gels such as DavisilSilica, E. Merck Silica Gel, Sigma-Aldrich Silica Gel (all availablefrom Suppleco) in addition to a wide range of silica gels available forvarious purposes as described in the Aldrich catalogue/handbook (AldrichChemical Company (Milwaukee, Wis.)). Preferred gel materials includeagarose based gels, various forms of acrylamide based gels (reagentsavailable from, e.g., Suppleco, SIGMA, Aldrich, SIGMA-Aldrich and manyother sources) colloidial solutions such as protein colloids (gelatins)and hydrated starches. Various forms of gels are discussed furtherbelow.

[0050] A variety of affinity media for purification and separation ofmolecular components are also available, including a variety of modifiedsilica gels available from SIGMA, Aldrich and SIGMA-Aldrich, as well asSuppleco, such as acrylic beads, agarose beads, cellulose, sepharose,sepharose CL, toyopearl or the like chemically linked to an affinityligand such as a biological molecule. A wide variety of activatedmatrixes, amino acid resins, avidin and biotin resins, carbohydrateresins, dye resins, glutathione resins, hydrophobic resins,immunochemical resins, lectin resins, nucleotide/coenzyme resins,nucleic acid resins, and specialty resins are available, e.g., fromSuppleco, SIGMA, Aldrich or the like. See also, Hermanson et al. (1992)Immobilized Affinity Ligand Techniques Academic Press.

[0051] Other media commonly used in chromatography are also adaptable tothe present invention, including activated aluminas, carbopacks,carbosieves, carbowaxes, chromosils, DEGS, Dexsil, Durapak, MolecularSieve, OV phases, pourous silica, chromosorb series packs, HayeSepseries, Porapak series, SE-30, Silica Gel, SP-1000, SP-1200, SP-2100,SP-2250, SP-2300, SP2401, Tenax, TCEP, supelcosil LC-18-S and LC-18-T,Methacrylate/DVBm, polyvinylalcohols, napthylureas, non-polar methylsilicone, methylpolysiloxane, poly(ethylene glycol) biscyanopropylpolysiloxane and the like.

[0052] Several methods of providing fluidic regions in selected regionsof a channel, or selected channels are provided. In a first aspect,multiple microfluidic regions are filled with a first fluid such as anunpolymerized solution that, upon polymerization, forms a sievingmatrix. Elements of the microfluidic device such as microfluidicchannels are filled with the first fluid by forcing the fluid into thechannel under pressure, or by moving the fluid into the channelelectrokinetically. In one embodiment, the first fluid polymerizes uponexposure to light (i.e., the fluid comprises a “photopolymerizable”polymer). The fluid is then selectively exposed to light (e.g., usingphotomasking techniques) in those regions where a polymerized gel isdesired. Unpolymerized fluid is then optionally washed out of theunselected regions of the microfluidic device, or into a waste reservoirusing electrokinetic flow or pressure.

[0053] A wide variety of free-radically polymerizable monomersphotopolymerize to form gels, or can be made photopolymerizeable by theaddition of, e.g., energy transfer dyes. For example, free-radicallypolymerizable monomers can be selected from acrylate, methacrylate andvinyl ester functionalized materials. They can be monomers and/oroligomers such as (meth)acrylates (meth)acrylamides, acrylamides, vinylpyrrolidone and azalactones. Such monomers include mono-, di-, orpoly-acrylates and methacrylates such as methyl acrylate, methylmethacrylate, ethyl acrylate, isopropyl methacrylate, isooctyl acrylate,isobornyl acrylate, isobornyl methacrylate, acrylic acid, n-hexylacrylate, stearyl acrylate, allyl acrylate, glycerol diacrylate,glycerol triacrylate, ethylene glycol diacrylate, diethyleneglycoldiacrylate, triethyleneglycol dimethacrylate, 1,6-hexanediol diacrylate,1,3-propanediol diacrylate, 1,3-propanediol dimethacrylate, trimethanoltriacrylate, 1,2,4-butanetriol trimethylacrylate, 1,4-cyclohexanedioldiacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate,pentaerythritol tetramethacrylate, sorbitol hexacrylate,bis[1-(2-acryloxy)]-p-ethoxyphenyl-dimethylmethane,bis[1-(3-acryloxy-2-hydroxy)]-propoxyphenyl dimethylmethane,tris-hydroxyethyl isocyanurate trimethacrylate; the bis-acrylates andbis-methacrylates of polyethylene glycols of molecular weight 200-500,copolymerizable mixtures of acrylated monomers, acrylated oligomers, PEGdiacrylates, etc. Strongly polar monomers such as acrylic acid,acrylamide, itaconic acid, hydroxyalkyl acrylates, or substitutedacrylamides or moderately polar monomers such as N-vinyl-2-pyrrolidone,N-vinyl caprolactam, and acrylonitrile are useful.

[0054] Proteins such as gelatin, collagen, elastin, zein, and albumin,whether produced from natural or recombinant sources, which are madefree-radical polymerization by the addition of carbon-carbon double ortriple bond-containing moieties, including acrylate, diacrylate,methacrylate, ethacrylate, 2-phenyl acrylate, 2-chloro acrylate, 2-bromoacrylate, itaconate, oliogoacrylate, dimethacrylate, oligomethacrylate,acrylamide, methacrylamide, styrene groups, and other biologicallyacceptable photopolymerizable groups, can also be used to form sievingmatrixes.

[0055] Dye-sensitized polymerization is well known in the chemicalliterature. For example, light from an argon ion laser (514 nm), in thepresence of an xanthin dye and an electron donor, such astriethanolamine, to catalyze initiation, serves to induce a free radicalpolymerization of acrylic groups in a reaction mixture (Neckers, et al.,(1989) Polym. Materials Sci. Eng., 60:15; Fouassier, et al., (1991)Makromol. Chem., 192:245-260). After absorbing laser light, the dye isexcited to a triplet state. The triplet state reacts with a tertiaryamine such as the triethanolamine, producing a free radical whichinitiates a polymerization reaction. Polymerization is extremely rapidand is dependent on the functionality of the composition, itsconcentration, light intensity, and the concentration of dye and, e.g.,amine.

[0056] Dyes can be used which absorb light having a frequency between320 nm and 900 nm, can form free radicals, are water soluble, etc. Thereare a large number of photosensitive dyes that can be used to opticallyinitiate polymerization, such as ethyl eosin, eosin Y, fluorescein,2,2-dimethoxy-2-phenyl acetophenone, 2-methoxy, 2-phenylacetophenone,camphorquinone, rose bengal, methylene blue, erythrosin, phloxime,thionine, riboflavin, methylene green, acridine orange, xanthine dye,and thioxanthine dyes.

[0057] Cocatalysts useful with photoinitiating dyes are typicallynitrogen based compounds capable of stimulating a free radical reaction.Primary, secondary, tertiary or quaternary amines are suitablecocatalysts, as are nitrogen atom containing electron-rich molecules.Cocatalysts include triethanolamine, triethylamine, ethanolamine,N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amine,N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethylethylenediamine, potassium persulfate, tetramethyl ethylenediamine,lysine, ornithine, histidine and arginine. Examples of thedye/photoinitiator system include ethyl eosin with an amine, eosin Ywith an amine, 2,2-dimethoxy-2-phenoxyacetophenone,2-methoxy-2-phenoxyacetophenone, camphorquinone with an amine, and rosebengal with an amine.

[0058] In some cases, dye may absorb light and initiate polymerization,without any additional initiator such as the amine. In these cases, onlythe dye and a monomer need be present to initiate polymerization uponexposure to light. The generation of free radicals is terminated whenthe laser light is removed. Some photoinitiators, such as2,2-dimethoxy-2-phenylacetophenone, do not require any auxiliary amineto induce photopolymerization; in these cases, the presence of dye,monomer and an appropriate wavelength light is sufficient forphotopolymerization.

[0059] Preferred light sources include various lamps and lasers such asthose which have a wavelength of about 320-800 nm. This light can beprovided by any appropriate source able to generate the desiredradiation, such as a mercury lamp, longwave UV lamp, He—Ne laser, anargon ion laser, etc. In a preferred embodiment, a UV source is used topolymerize a UV photopolymerizeable gel. Similarly, the light sourceused is typically selected based upon the chemistry which is to beaffected by the source.

[0060] Similarly, a variety of gels can be selectively polymerized byexposure to heat. As described herein, selective heat control usingapplied current is easily performed in the microfluidic apparatus of theinvention, providing for simplified control of gel polymerizationthrough thermal processes. Examples include initiation by thermalinitiators, which form free radicals at moderate temperatures, such asbenzoyl peroxide, with or without triethanolamine, potassium persulfate,with or without tetramethylethylenediamine, and ammonium persulfate withsodium bisulfite.

[0061] For either the thermal or photopolymerization methods herein,monomer is pumped, e.g., in aqueous buffer, into a channel or channelregion using electroosmotic flow, or using a pressure gradient. Afterselective exposure to light or heat, as appropriate, unpolymerizedmaterials are removed, typically using electroosmotic flow, butoptionally using a pressure gradient, from regions where monomermaterial is undesirable.

[0062] In another embodiment, the first fluid is polymerized byselectively exposing certain channel regions to an activator orcross-linker. For example, where the fluid is polyacrylamide, theactivator/cross linker can be TEMED and APS. In this embodiment, thereagents are placed into a well and electrokinetically loaded intoselected channel regions of a microfluidic substrate. After selectiveexposure to activator/cross linker as appropriate, unpolymerizedmaterials are removed from regions where monomer material isundesirable, typically using electroosmotic flow (but optionally using apressure gradient). Often the material will be shunted to one or morewaste buffer where the material is optionally removed, e.g., bypipetting or electropipeting the material out of the well.

[0063] In another embodiment, a sieving matrix is deposited throughout achannel or channels of a microfluidic device in a form which is subjectto electroosmosis (i.e., the matrix moves electrokinetically in thechannel). The matrix is then selectively replaced by a second fluidicphase (e.g., a buffer) in selected regions of a channel byelectrokinetically loading the buffer in the selected region.

[0064] In an additional embodiment, a first fluidic phase is loaded intomultiple channels of a microfluidic device and polymerized in place.Selective components which solubilize the polymerized gel are thenloaded (e.g., electrokinetically or under pressure) into channel regionswhere polymerized product is not desired. The selected componentsdissolve the polymerized gel. Example of solubilization compoundsinclude acids, bases and other chemicals. In one preferred embodiment,at least two compounds are used to dissolve polymerized products, whereboth products need to be present to dissolve the polymer. This providesfor fine control of dissolution, e.g., where each chemical is underseparate electrokinetic control. An example of such a chemical pair isDTT(N,N′-bis(acrylol)cystamine or (1,2-dihydroxyethylene-bis-acrylamide)[DHEBA] and sodium periodiate or calcium alginate+EDTA or TCEP-HCL andN,N′-bis(acryloyl)cystamine. A variety of such materials are known.

Microfluidic Apparatus Applications—PCR and In Vitro Amplification

[0065] The multi-phasic apparatus of the invention are particularlyuseful for performing experimental or diagnostic procedures whichcombine fluid mixing and product separatory aspects. For example, in afirst fluidic phase, reactions such as PCR are performed, followed byseparation in a sieving matrix in different channel region. Bench scalein vitro amplification techniques suitable for amplifying sequences toprovide a nucleic acid e.g., as a diagnostic indicator for the presenceof the sequence, or for subsequent analysis, sequencing or subcloningare known.

[0066] In brief, the most common form of in vitro amplification, i.e.,PCR amplification, generally involves the use of one strand of thetarget nucleic acid sequence as a template for producing a large numberof complements to that sequence. As used herein, the phrase “targetnucleic acid sequence” generally refers to a nucleic acid sequence, orportion of a nucleic acid sequence that is the subject of a particularfluidic operation, e.g., analysis, amplification, identification or thelike. Generally, two primer sequences complementary to different ends ofa segment of the complementary strands of the target sequence hybridizewith their respective strands of the target sequence, and in thepresence of polymerase enzymes and nucleoside triphosphates, the primersare extended along the target sequence through the action of thepolymerase enzyme (in asymmetric PCR protocols, a single primer isused). The extensions are melted from the target sequence by raising thetemperature of the reaction mixture, and the process is repeated, thistime with the additional copies of the target sequence synthesized inthe preceding steps. PCR amplification typically involves repeatedcycles of denaturation, hybridization and extension reactions to producesufficient amounts of the target nucleic acid, all of which are carriedout at different temperatures. Typically, melting of the strands, orheat denaturation, involves temperatures ranging from about 90° C. to100° C. for times ranging from seconds to minutes. The temperature isthen cycled down, e.g., to between about 40° C. and 65° C. forannealing, and then cycled up to between about 70° C. and 85° C. forextension of the primers along the target strand.

[0067] Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods at benchtop scales, including thepolymerase chain reaction (PCR) the ligase chain reaction (LCR),Qβ-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well asMullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al. eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringeret al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology13: 563-564. Improved methods of cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids by PCR are summarized in Chenget al. (1994) Nature 369: 684-685 and the references therein, in whichPCR amplicons of up to 40 kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase. See, Ausbel, Sambrook andBerger, all supra.

[0068] The use of PCR for amplifying nucleic acids is ubiquitous inmolecular biology for the amplification and detection of nucleic acids.PCR technologies are well-suited to forensic analysis, paternitytesting, maternity testing, infectious disease diagnosis (e.g.,detection of a nucleic acid from a pathogenic organism such as a virus,e.g., HIV, HTLV, herpes virus, etc.), cancer diagnosis (e.g., for thedetection of oncogenic gene products such as p53 nucleic acids, Her 2nucleic acids, etc.). Use of PCR for these techniques is well known andadaptable to use with the present invention.

[0069] In one aspect, PCR or other thermal reaction reagents (e.g., LCRreagents) such as thermostable polymerase, DNA template, primers,nucleotides and buffers are mixed in a microchannel or chamber, with theentire microfluidic substrate (e.g., a LABCHIP™ from CaliperTechnologies) being subject to repeated cycles of heating and cooling,e.g., on a thermocycler or by switching between a hot plate and a heatsink.

[0070] In a second more preferred embodiment, variations in channelthickness and/or voltage are used selectively to heat selected regionsof a channel which contain a PCR reaction. PCR amplification isparticularly well suited to this use in the apparatus, methods andsystems of the invention. Thermocycling amplification methods, includingPCR and LCR, are conveniently performed in microscale devices, makingiterative fluidic operations involving PCR well suited to use in methodsand devices of the present invention (see also, U.S. Pat. Nos. 5,498,392and 5,587,128 to Willingham et al.). Accordingly, in one preferredembodiment, generation of amplicons such as sequencing templates usingPCR, or direct sequencing of nucleic acids by PCR (e.g., using nucleasedigestion as described supra) is performed with the integrated systemsand devices of the invention.

[0071] Thermocycling in microscale devices, including thermocycling byjoule heating is described in co-pending application U.S. S No.60/056058, attorney docket number 017646-003800 entitled “ELECTRICALCURRENT FOR CONTROLLING FLUID TEMPERATURES IN MICROCHANNELS” filed Sep.2, 1997 by Calvin Chow, Anne R. Kopf-Sill and J. Wallace Parce; in Ser.No. 08/977,528, filed Nov. 25, 1997; provisional patent application U.S.S No. 60/068,311, entitled “Closed Loop Biochemical Analyzer” by Knapp,filed Dec. 19, 1997 and in CLOSED-LOOP BIOCHEMICAL ANALYZERS, attorneydocket number 017646-003100 filed Apr. 3, 1998. In brief, energy isprovided to heat fluids, e.g., samples, analytes, buffers and reagents,in desired locations of the substrates in an efficient manner byapplication of electric current to fluids in microchannels. Thus, thepresent invention optionally uses power sources that pass electricalcurrent through a first fluidic phase in a first channel region forheating purposes, as well as for material transport. In exemplaryembodiments, the fluid of the first fluidic phase passes through achannel of a desired cross-section (e.g., diameter) to enhance thermaltransfer of energy from the current to the fluid. The channels can beformed on almost any type of substrate material such as, for example,amorphous materials (e.g., glass, plastic, silicon), composites,multi-layered materials, combinations thereof, and the like.

[0072] In general, electric current passing through the fluid in achannel produces heat by dissipating energy through the electricalresistance of the fluid. Power dissipates as the current passes throughthe fluid and goes into the fluid as energy as a function of time toheat the fluid. The following mathematical expression generallydescribes a relationship between power, electrical current, and fluidresistance:

POWER=P ² R

[0073] where

[0074] POWER=power dissipated in fluid;

[0075] I=electric current passing through fluid; and

[0076] R=electric resistance of fluid.

[0077] The above equation provides a relationship between powerdissipated (“POWER”) to current (“I”) and resistance (“R”). In some ofthe embodiments, which are directed toward moving fluid in channels,e.g., to provide mixing, electrophoretic separation, or the like, aportion of the power goes into kinetic energy of moving the fluidthrough the channel. However, it is also possible to use a selectedportion of the power to controllably heat fluid in a channel or selectedchannel regions. A channel region suitable for heating is often narroweror smaller in cross-section than other channel regions in the channelstructure, as a smaller cross-section provides higher resistance in thefluid, which increases the temperature of the fluid as electric currentpasses through. Alternatively, the electric current is increased acrossthe length of the channel by increased voltage, which also increases theamount of power dissipated into the fluid to correspondingly increasefluid temperature.

[0078] To selectively control the temperature of fluid at a region ofthe channel, a power supply applies voltage and/or current in one ofmany ways. For instance, a power supply can apply direct current (i.e.,DC) or alternating current (AC), which passes through the channel andinto a channel region which is smaller in cross-section, thereby heatingfluid in the region. This current is selectively adjusted in magnitudeto complement any voltage or electric field that is applied to movefluid in and out of the region. AC current, voltage, and/or frequencycan be adjusted, for example, to heat the fluid without substantiallymoving the fluid. Alternatively, a power supply can apply a pulse orimpulse of current and/or voltage, which passes through the channel andinto a channel region to heat fluid in the region. This pulse isselectively adjusted to complement any voltage or electric field that isapplied to move fluid in and out of the region. Pulse width, shape,and/or intensity can be adjusted, for example, to heat the fluidsubstantially without moving the fluid or to heat the fluid while movingthe fluid. Still further, the power supply can apply any combination ofDC, AC, and pulse, depending upon the application. In practice, directapplication of electric current to fluids in the microchannels of theinvention results in extremely rapid and easily controlled changes intemperature.

[0079] A controller or computer such as a personal computer monitors thetemperature of the fluid in the region of the channel where the fluid isheated. The controller or computer receives current and voltageinformation from, for example, the power supply and identifies ordetects temperature of fluid in the region of the channel. Dependingupon the desired temperature of fluid in the region, the controller orcomputer adjusts voltage and/or current to meet the desired fluidtemperature. The controller or computer also can be set to be “currentcontrolled” or “voltage controlled” or “power controlled” depending uponthe application.

[0080] The region which is heated can be a “coil” which is optionally ina planar arrangement. Transfer of heat from the coil to a reactionchannel through a substrate material is used to heat the reactionchannel. Alternatively, the coil itself is optionally the reactionchannel.

[0081] A voltage is applied between regions of the coil to directcurrent through the fluid for heating purposes. In particular, a powersupply provides a voltage differential between regions of the coil.Current flows between the regions and traverses a plurality of coils orcoil loops (which can be planar), which are defined by a substrate.Shape and size of the coils can influence an ability of current to heatthe fluid in the coil. As current traverses through the fluid, energy istransferred to the fluid for heating purposes. Cooling coils can also beused. As a cooling coil, a fluid traverses from region to region in thecoil, which can be placed to permit heat transfer through a substratefrom a sample. The cooling fluid can be a variety of substancesincluding liquids and gases. As merely an example, the cooling fluidincludes aqueous solutions, liquid or gaseous nitrogen, and others. Thecooling fluid can be moved between regions using any of the techniquesdescribed herein, and others. Further details are found in Chow et al.,supra.

[0082] The introduction of electrical current into fluid causes heat(this procedure is referred to as “Joule heating”). In the examples offluid movement herein where thermal effects are not desired, the heatingeffect is minimal because, at the small currents employed, heat israpidly dissipated into the chip itself. By substantially increasing thecurrent across the channel, rapid temperature changes are induced thatcan be monitored by conductivity. At the same time, the fluid can bekept static in the channel by using alternating instead of directcurrent. Because nanoliter volumes of fluid have tiny thermal mass,transitions between temperatures can be extremely short. Oscillationsbetween any two temperatures above 0° C. and below 100° C. in 100milliseconds have been performed.

[0083] Joule heating in microchannels is an example of how benchtopmethods can be dramatically improved in the formats provided herein. PCRtakes hours to perform currently, primarily because it takes a long timefor conventional heating blocks to oscillate between temperatures. Inaddition, reagent cost is an obstacle to massive experimentation. Boththese parameters are altered by orders of magnitude in the LabChip™format.

[0084] In one aspect, PCR reaction conditions are controlled as afunction of channel geometry. Microfabrication methods permit themanufacture of channels that have precise variations in cross sectionalarea. Since the channel resistance is inversely proportional to thecross sectional area, the temperature varies with the width and depth ofthe channel for a given flow of current. As fluid moves through astructure of varying cross sectional area, its temperature will change,depending on the dimensions of the channel at any given point. Theamount of time it experiences a given temperature will be determined bythe velocity of the fluid flow, and the length of channel with thosedimensions. This concept is illustrated in FIG. 2. Nucleic acids oftypical lengths have a low diffusion coefficient (about 10⁻⁷ cm/sec²).Thus over the time frame necessary to affect amplification, DNA willonly diffuse a few hundred microns. In a given channel, reactions of afew nanoliters will occupy a few millimeters. Thus in devices ofconvenient length (a few centimeters), many PCR reactions can beperformed concurrently yielding new amplification products every fewseconds per channel. In parallel formats, hundreds of separate reactionscan be performed simultaneously. Because of its simplicity, throughputand convenience, this amplification unit is a preferred feature of manyassays herein.

[0085] In FIG. 2, amplification reactions are performed concurrently inseries using biased alternating current to heat the fluid inside thechannel and move material through it. The time for each step of thereaction is controlled by determining the speed of movement and thelength of channel having particular widths. Flow can be reversed toallow a single small channel region to be used for many separateamplifications.

[0086] As depicted, several samples are run simultaneously in channel210. Sample 215 is in narrow channel region 220; in operation, thisregion is heated to, e.g., 95° C. (hot enough to denature nucleic acidspresent in sample 215, but cool enough that thermostable reagents suchas Taq DNA polymerase are relatively stable due to the relative size ofregion 220 and the applied current. Concurrently, wide channel region230 is heated, e.g., to 60° C. (cool enough for binding of primers insample 225 and initiation of polymerase extension), due to the relativesize of region 230 and the applied current. Concurrently, intermediatechannel region 235 is heated, e.g., to 72° C. (hot enough to preventunwanted non-specific primer-target nucleic acid interactions in sample240 and cool enough to permit continued polymerase extension), due tothe relative size of region 235 and the applied current. This processcan be concurrently carried out with a plurality of additional channelregions such as narrow region 245, wide region 250 and intermediateregion 255, with samples 260, 265 and 270.

[0087] Where possible, direct detection of amplified products can beemployed. For example, differentially labeled competitive probehybridization is used for single nucleotide discrimination.Alternatively, molecular beacons or single nucleotide polymeraseextension can be employed. Homogeneous detection by fluorescencepolarization spectroscopy can also be utilized (fluorescencepolarization has been used to distinguish between labeled smallmolecules free in solution or bound to protein receptors).

PCR Chips

[0088] One aspect of the present invention is the surprising discoverythat PCR can be performed in the presence of sieving matrix, and thatthe products of the PCR reaction are separable in the matrix in amicrofludic channel (see also, the examples below).

[0089] Accordingly, in one aspect, the invention provides new method ofperforming PCR. In the methods, components of a PCR reaction mixture(i.e., the molecules which participate in a PCR reaction, such as PCRextension primers, nucleotide triphosphates, thermostable enzymes, ionsand buffer components such as Mg++, template DNAs, etc.) are mixed witha sieving matrix to provide a PCR sieving matrix mixture. The resultingmixture is then repetitively thermocycled as described supra to produceone or more PCR products.

[0090] In preferred embodiments, the components of the PCR reactionmixture are mixed with the sieving matrix in a microfluidic channel,e.g., a channel on a LABCHIP™. The apparatus can include one or moreadditional channels crossing the microfluidic channel and optionallyincludes fluid (or joule heating) means such as an electrokineticcontroller. Detection regions in the channels, and correspondingdetectors are also useful. The PCR products are typicallyelectrophoresed through the channels to achieve product separation.

[0091] It will be appreciated that separations chips comprising a singlematrix separations phase are produced as described above, thus, for thisembodiment, multiple fluidic phases in the apparatus are not necessary.However, additional fluidic phases can be placed in additional channelsor channel regions in fluid communication with a channel regioncomprising the PCR sieving mixture for electrophoretic or electroosmoticmovement of the PCR components or products in the chips. For example, insome aspects a PCR reaction product is selected for furthermanipulations such as cloning, sequencing or the like, all of which areperformed in PCR chips (see also, U.S. S No. 60/068,311, entitled“Closed Loop Biochemical Analyzer” by Knapp, filed Dec. 19, 1997 and“Closed Loop Biochemical Analyzers” by Knapp et al. U.S. Ser. No. ______(attorney docket number 017646-0003100US) filed Apr. 3, 1998).

Microfluidic Apparatus Applications—DNA Sequencing

[0092] The multiphasic devices and methods of the present invention arealso particularly applicable to nucleic acid sequencing. It will beappreciated that DNA sequencing reactions can be run in a first fluidphase of the apparatus, with separatory steps being performed in asieving matrix.

[0093] The integrated systems of the invention are useful for sequencinga wide variety of nucleic acid constructs. Essentially any DNA templatecan be sequenced, with the selection of the nucleic acid to be sequenceddepending upon the construct in hand by the sequencer. Thus, an initialstep in the methods of the invention is the selection or production of atemplate nucleic acid to be sequenced.

[0094] Many methods of making recombinant ribo and deoxyribo nucleicacids, including recombinant plasmids, recombinant lambda phage,cosmids, yeast artificial chromosomes (YACs), P1 artificial chromosomes,Bacterial Artificial Chromosomes (BACs), and the like are known. Thesequencing of large nucleic acid templates is advantageously performedby the present methods, systems and apparatus, because an entire nucleicacid can be sequenced by primer walking along the length of the templatein several rapid cycles of sequencing.

[0095] Examples of appropriate cloning techniques for making nucleicacids, and instructions sufficient to direct persons of skill throughmost standard cloning and other template preparation exercises are foundin Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.)Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,(Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1997, supplement 37)(Ausubel). Basic procedures for cloning and other aspects of molecularbiology and underlying theoretical considerations are also found inLewin (1995) Genes V Oxford University Press Inc., NY (Lewin); andWatson et al. (1992) Recombinant DNA Second Edition Scientific AmericanBooks, NY. Product information from manufacturers of biological reagentsand experimental equipment also provide information useful in knownbiological methods. Such manufacturers include the Sigma ChemicalCompany (Saint Louis, Mo.); New England Biolabs (Beverly, Mass.); R&Dsystems (Minneapolis, Minn.); Pharmacia LKB Biotechnology (Piscataway,N.J.); CLONTECH Laboratories, Inc. (Palo Alto, Calif.); ChemGenes Corp.,(Waltham Mass.) Aldrich Chemical Company (Milwaukee, Wis.); GlenResearch, Inc. (Sterling, Va.); GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.); Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland); Invitrogen (San Diego, Calif.); Perkin Elmer(Foster City, Calif.); and Strategene; as well as many other commercialsources known to one of skill.

[0096] In one aspect, the generation of large nucleic acids is useful inpracticing the invention. It will be appreciated that such templates areparticularly useful in some aspects where the methods and devices of theinvention are used to sequence large regions of DNA, e.g., for genomicstypes of applications. An introduction to large clones such as YACs,BACs, PACs and MACs as artificial chromosomes is provided by Monaco andLarin (1994) Trends Biotechnol 12 (7): 280-286.

[0097] The construction of nucleic acid libraries of template nucleicacids is described in the above references. YACs and YAC libraries arefurther described in Burke et al. (1987) Science 236:806-812. Griddedlibraries of YACs are described in Anand et al. (1989) Nucleic AcidsRes. 17, 3425-3433, and Anand et al. (1990) Nucleic Acids Res. Riley(1990) 18:1951-1956 Nucleic Acids Res. 18(10): 2887-2890 and thereferences therein describe cloning of YACs and the use of vectorettesin conjunction with YACs. See also, Ausubel, chapter 13. Cosmid cloningis also well known. See, e.g., Ausubel, chapter 1.10.11 (supplement 13)and the references therein. See also, Ish-Horowitz and Burke (1981)Nucleic Acids Res. 9:2989-2998; Murray (1983) Phage Lambda and MolecularCloning in Lambda II (Hendrix et al., eds) 395-432 Cold Spring HarborLaboratory, NY; Frischauf et al. (1983) J. Mol. Biol. 170:827-842; and,Dunn and Blattner (1987) Nucleic Acids Res. 15:2677-2698, and thereferences cited therein. Construction of BAC and P1 libraries is wellknown; see, e.g., Ashworth et al. (1995) Anal Biochem 224 (2): 564-571;Wang et al. (1994) Genomics 24(3): 527-534; Kim et al. (1994) Genomics22(2): 336-9; Rouquier et al. (1994) Anal Biochem 217(2): 205-9; Shizuyaet al. (1992) Proc Natl Acad Sci USA 89(18): 8794-7; Kim et al. (1994)Genomics 22 (2): 336-9; Woo et al. (1994) Nucleic Acids Res 22(23):4922-31; Wang et al. (1995) Plant (3): 525-33; Cai (1995) Genomics 29(2): 413-25; Schmitt et al. (1996) Genomics 1996 33(1): 9-20; Kim et al.(1996) Genomics 34(2): 213-8; Kim et al. (1996) Proc Natl Acad Sci USA(13): 6297-301; Pusch et al. (1996) Gene 183(1-2): 29-33; and, Wang etal. (1996) Genome Res 6(7): 612-9. In general, where the desired goal ofa sequencing project is the sequencing of a genome or expression profileof an organism, a library of the organism's cDNA or genomic DNA is madeaccording to standard procedures described, e.g., in the referencesabove. Individual clones are isolated and sequenced, and overlappingsequence information is ordered to provide the sequence of the organism.See also, Tomb et al. (1997) Nature 539-547 describing the whole genomerandom sequencing and assembly of the complete genomic sequence ofHelicobacter pylori; Fleischmann et al. (1995) Science 269:496-512describing whole genome random sequencing and assembly of the completeHaemophilus influenzae genome; Fraser et al. (1995) Science 270:397-403describing whole genome random sequencing and assembly of the completeMycoplasma genitalium genome and Bult et al. (1996) Science273:1058-1073 describing whole genome random sequencing and assembly ofthe complete Methanococcus jannaschii genome.

[0098] The nucleic acids sequenced by this invention, whether RNA, cDNA,genomic DNA, or a hybrid of the various combinations, are isolated frombiological sources or synthesized in vitro. The nucleic acids of theinvention are present in transformed or transfected whole cells, intransformed or transfected cell lysates, or in a partially purified orsubstantially pure form.

[0099] In a preferred aspect, the invention provides a multi-phasic“closed loop” device for determining the entire sequence of an unknownDNA molecule of interest by iteratively sequencing sub regions of themolecule of interest and compiling the subsequence information. Closedloop sequencing strategies are described in provisional patentapplication U.S. S No. 60/068,311, entitled “Closed Loop BiochemicalAnalyzer” by Knapp, filed Dec. 19, 1997 and “Closed Loop BiochemicalAnalyzers” by Knapp et al. U.S. Ser. No. ______ (attorney docket number017646-0003100US) filed Apr. 3, 1998.

[0100] In brief, oligonucleotide primers are chosen from a pool ofpossible sequencing primers upon determination of an initial portion ofthe DNA sequence. With iterative utilization of this strategy, it ispossible to primer walk through an entire sequence without synthesizingnew primers. A template nucleic acid is selected and introduced into areaction channel in a microfluidic (generally electroosmotic) device ofthe invention. The template is optionally amplified, e.g., byintroducing PCR or LCR reagents into the channel and performing cyclesof heating and cooling on the template. Alternatively, e.g., where thesource of template is from an abundant sequence such as a cloned nucleicacid, further amplification can be unnecessary. In addition toamplification procedures, a PCR nuclease chain termination procedure canalso be used for direct sequencing in the methods of the invention.Porter et al. (1997) Nucleic Acids Research 25(8):1611-1617 describe thebiochemistry of PCR chain termination methods.

[0101] Sequencing reagents are added to the template nucleic acid and asequencing reaction is performed appropriate to the particular reactionin use. Many appropriate reactions are known, with the Sanger dideoxychain termination method being the most common. See, Sanger et al.(1977) Proc. Nat. Acad. Sci., USA 74:5463-5467. The primer used to primesynthesis is typically selected from a pre-synthesized set of nucleicacid primers, preferably a set including many or all of the primers fora particular primer length. In a preferred aspect, modular primers areused. For modular primer strategies, see, Beskin et al. (1995) NucleicAcids Research 23(15):2881-2885). A modified version of the use of themodular primer strategy, in which small nucleotide primers arespecifically elongated for use in PCR to amplify and sequence templatenucleic acids has also been described. The procedure is referred to asDNA sequencing using differential extension with nucleotide subsets(DENS). See, Raja et al. (1997) Nucleic Acids Research 25(4):800-805.

[0102] After the sequencing reaction is run, the products are separatedby size and/or charge in an analysis region of the microfluidic devicewhich comprises a second fluidic phase adapted for separation of nucleicacids, such as a sieving matrix as discussed above. As discussed herein,the devices of the invention can be used to electrophoretically separatemacromolecules by size and/or charge. The separated products aredetected, often as they pass a detector (nucleic acids are typicallylabeled with radioactive nucleotides or fluorophores; accordinglyappropriate detectors include spectrophotometers, fluorescent detectors,microscopes (e.g., for fluorescent microscopy) and scintillationcounting devices). Detection of size separated products is used tocompile sequence information for the region being sequenced. A computeris typically used to select a second primer from the pre-synthesizedprimer set which hybridizes to the sequenced region, and the process isiteratively repeated with the second primer, leading to sequencing of asecond region, selection of a third primer hybridizing to the secondregion, etc.

[0103] Selecting and Making Primers for Sequencing and PCR

[0104] Oligonucleotides for use as primers or probes, e.g., insequencing or PCR or non-thermal amplification reactions in microfluidicapparatus are typically synthesized chemically according to the solidphase phosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using anautomated synthesizer, as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custommade and ordered from a variety of commercial sources known to personsof skill. Purification of oligonucleotides, where necessary, istypically performed by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier (1983) J.Chrom. 255:137-149. The sequence of the synthetic oligonucleotides canbe verified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology 65:499-560.

[0105] While primers can hybridize to any of a number of sequences,selecting optimal primers is typically done using computer assistedconsideration of available sequences and excluding potential primerswhich do not have desired hybridization characteristics, and/orincluding potential primers which meet selected hybridizationcharacteristics. This is done by determining all possible nucleic acidprimers, or a subset of all possible primers with selected hybridizationproperties (e.g., those with a selected length, G:C ratio, uniqueness inthe given sequence, etc.) based upon the known sequence. The selectionof the hybridization properties of the primer is dependent on thedesired hybridization and discrimination properties of the primer. Ingeneral, the longer the primer, the higher the melting temperature. Inaddition, it is more difficult to generate a set of primers whichincludes all possible oligonucleotides for a given length, as therequired number of primers increases exponentially. For example, allpossible 3-mers requires 4³ primers, all possible 4-mers requires 4⁴primers, all possible 5-mers requires 4⁵ primers, all possible 6-mersrequires 4⁶ primers, etc. Standard sequencing primers are often in therange of 15-20 nucleotides in length, which would require sets of 4¹⁵ to4²⁰ nucleotides, or 1.1×10⁹ to 1.1×10¹² primers.

[0106] While it is possible to make such large sets of primers usingcombinatorial chemical techniques, the associated problems of storingand retrieving billions or even trillions of primers make these primersets less desirable. Instead, smaller sets of primers used in a modularfashion are desirable.

[0107] For example, Ulanovsky and co-workers have described themechanism of the modular primer effect (Beskin et al. (1995) NucleicAcids Research 23(15):2881-2885) in which short primers of 5-6nucleotides can specifically prime a template-dependent polymeraseenzyme when 2-3 contiguously annealing, but unligated, primers are usedin a polymerase dependent reaction such as a sequencing reaction.Polymerase enzymes are preferentially engaged by longer primers, whethermodular or conventional, accounting for the increased specificity ofmodular primers. Because it is possible to synthesize easily allpossible primers with 5-6 nucleotides (i.e., 4⁵ to 4⁶ or 1024 to 4096primers), it is possible to generate and utilize a universal set ofnucleotide primers, thereby eliminating the need to synthesizeparticular primers to extend nucleotide sequencing reactions ofnucleotide templates. In an alternative embodiment, a ligase enzyme isused to ligate primers which hybridize to adjacent portions of atemplate, thereby providing a longer primer.

[0108] A modified version of the use of the modular primer strategy, inwhich small nucleotide primers are specifically elongated for use in PCRto amplify and sequence template nucleic acids has also been described.The procedure is referred to as DNA sequencing using differentialextension with nucleotide subsets (DENS). See, Raja et al. (1997)Nucleic Acids Research 25(4):800-805. Thus, whether standardSanger-style sequencing or direct PCR sequencing using boronatednucleotides and a nuclease (see, Porter et al. 1997, supra.) aredesired, small sets of short primers are sufficient for use insequencing and PCR and are desirable.

[0109] It is expected that one of skill is thoroughly familiar with thetheory and practice of nucleic acid hybridization and primer selection.Gait, ed. Oligonucleotide Synthesis: A Practical Approach, IRL Press,Oxford (1984); W. H. A. Kuijpers Nucleic Acids Research 18(17), 5197(1994); K. L. Dueholm J. Org. Chem. 59, 5767-5773 (1994); S. Agrawal(ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993)Laboratory Techniques in biochemistry and molecularbiology—hybridization with nucleic acid probes, e.g., part I chapter 2“overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. provide a basic guide to nucleic acidhybridization. Innis supra provides an overview of primer selection.

[0110] One of skill will recognize that the 3′ end of an amplificationprimer is more important for PCR than the 5′ end. Investigators havereported PCR products where only a few nucleotides at the 3′ end of anamplification primer were complementary to a DNA to be amplified. Inthis regard, nucleotides at the 5′ end of a primer can incorporatestructural features unrelated to the target nucleic acid; for instance,in one embodiment, a sequencing primer hybridization site (or acomplement to such as primer, depending on the application) isincorporated into the amplification primer, where the sequencing primeris derived from a primer used in a standard sequencing kit, such as oneusing a biotinylated or dye-labeled universal M13 or SP6 primer. Thesestructural features are referred to as constant primer regions. Theprimers are typically selected so that there is no complementaritybetween any known target sequence and any constant primer region. One ofskill will appreciate that constant regions in primer sequences areoptional.

[0111] The primers are selected so that no secondary structure formswithin the primer. Self-complementary primers have poor hybridizationproperties, because the complementary portions of the primers selfhybridize (i.e., form hairpin structures). Modular primers are selectedto have minimal cross-hybridization, thereby preventing competitionbetween individual primers and a template nucleic acid and preventingduplex formation of the primers in solution, and possible concatenationof the primers during PCR. If there is more than one constant region inthe primer, the constant regions of the primer are selected so that theydo not self-hybridize or form hairpin structures.

[0112] One of skill will recognize that there are a variety of possibleways of performing the above selection steps, and that variations on thesteps are appropriate. Most typically, selection steps are performedusing simple computer programs to perform the selection as outlinedabove; however, all of the steps are optionally performed manually. Oneavailable computer program for primer selection is the MacVector™program from Kodak. An alternate program is the MFOLD program (GeneticsComputer Group, Madison Wis.) which predicts secondary structure of,e.g., single-stranded nucleic acids. In addition to programs for primerselection, one of skill can easily design simple programs for any or allof the preferred selection steps.

Integrated Multiphasic Microfluidic Apparatus and Systems

[0113] Multiphasic analysis systems are provided, for example forperforming nucleic acids-based diagnostic and sequencing applications,e.g., in a reference laboratory setting. The system typically hasseveral components: a multi-phasic microfluidic specimen and reagenthandling system; an “operating system” for processing integratedmicrofluidic experimentation steps; application-specific analysisdevices (optionally referred in this application e.g., as “LabChips™”(LabChip™ is a trademark of Caliper Technologies, Corp., Palo AltoCalif.); a label detection system, and multiple software components thatallow the user to interact with the system, and run processing steps,interpret data, and report results.

[0114] The microfluidic apparatus of the invention typically comprise asubstrate in which fluidic reagents, mixtures of reagents, reactants,products or the like are mixed and analyzed, in channels comprisingdifferent fluidic phases. A wide variety of suitable substrates for usein the devices of the invention are described in U.S. Ser. No.08/761,575, entitled “High Throughput Screening Assay Systems inMicroscale Fluidic Devices” by Parce et al. A microfluidic substrateholder is optionally incorporated into the devices of the invention forholding and/or moving the substrate during an assay. The substrateholder optionally includes a substrate viewing region for analysis ofreactions carried out on the substrate. A label detector mountedproximal to the substrate viewing region to detect formation of productsand/or passage of reactants along a portion of the substrate isprovided. A computer, operably linked to the analyte detector, monitorsformation of reactants, separation of sequencing products, or the like.An electrokinetic component typically provides for movement of thefluids on the multiphasic substrate.

[0115] A principal component of nucleic acid analysis is molecularpartition, performed, e.g., in a separatory phase of the presentinvention. In addition, the dexterous fluidics in the microfluidicdevices herein produce exquisite control over injection volume—aparameter determining resolution in molecular partitioning (typically,in the multi-phasic apparatus of the invention, stacking of componentsat the interface between fluidic phases provides for enhancedresolution). Aside from biochemistry and analytical capabilities inmicrodevices, systems that automate access to reagents and specimens arehighly useful for the integrated multiphasic systems herein. In highthroughput pharmaceutical screening a “world-to-chip” interface capableof importing samples from conventional liquid vessels (such as testtubes or 384-well plates), or from solid dots of reagent on substratesis useful.

[0116] Accordingly, in one embodiment, a “sipping” strategy forintroducing solubilized reagents or samples into a microfluidicsubstrate from a standard microplate is used. This is adapted toelements of nucleic acids testing, for example to allow for randomaccess to a library of probes or primers.

[0117] In order to take advantage of the very small quantities ofreagents required by the chip, and to make a system scalable to millionsof experiments, a solid phase reagent interface uniquely suited to highthroughput LabChip processing is desirable. Several interfaces that makeuse of reagents dried in microarrays on a solid surface are described inU.S. S No. 60/068,311, entitled “Closed Loop Biochemical Analyzer” byKnapp, filed Dec. 19, 1997 and “Closed Loop Biochemical Analyzers” byKnapp et al. U.S. Ser. No. ______ (attorney docket number017646-0003100US) filed Apr. 3, 1998. These configurations are suited tothe needs of diagnostic products in which elements need to bestandardized, convenient, and have acceptable shelf-life.

[0118] In brief, many robotic systems are now available that can depositarrays of individual solutions at high densities (1000 per squarecentimeter and greater). These are typically used as capture elements inheterogeneous phase biochemical assays such as nucleic acidshybridization. The same approach can be used to deposit elements ofsolution phase reactions (PCR primers, probes, sequencing primers,etc.). Using these approaches, systems that access solid phase reagentsat densities of up to 1000 spots per square centimeter are made.

[0119] As described above, a preferred integrated method of theinvention incorporates the use of pre-synthesized sets of primers forsequencing and/or PCR, and or reagents to be tested in drug screeningassays. A device of the invention preferably includes a primer storageand/or primer transport mechanism for delivering selected primers to areaction channel in the microfluidic device. Exemplary storagemechanisms optionally include components adapted to holding primers in aliquid or lyophilized form, including containers, containers withseparate compartments, plates with wells (e.g., small microtiter plateswith hundreds or thousands of wells) membranes, matrices, arrays ofpolymers, or the like. Additional embodiments for handling driedreagents on solid substrates are shown below.

[0120] In one embodiment, the primer storage area is physicallyseparated from the substrate. In this embodiment, the primers can beloaded onto the substrate, either manually, or using an automatedsystem. For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.)automated robot using a Microlab 2200 (Hamilton; Reno, Nev.) pipettingstation can be used to transfer parallel samples to regularly spacedwells in a manner similar to transfer of samples to microtiter plates.If the primers are stored in lyophilized form (e.g., dried on asubstrate), a portion of the lyophilized primer is typically suspendedin an aqueous solution to facilitate transfer to a microfluidicsubstrate. An electropipettor can be used to select and transportsamples to a microfluidic substrate from a well plate, or from anyregion of a microfluidic substrate. Because integration of theelectropipettor with the microfluidic substrates of the invention isrelatively simple, electropipettor embodiments are preferred.

[0121] In preferred embodiments including an electropipettor, a varietyof storage systems for storing reagents, such as primers for delivery tothe microfluidic devices of the invention, are applicable. Compounds areconveniently sampled with the electropipettor from well plates, or fromimmobilized samples stored on a matrix (e.g., a porous, hydrophilic, orhydrophobic matrix), or from dried samples stored on a substrate such asa nitrocellulose, nylon or nytran membrane. In embodiments where thesamples are dried, the samples are solubilized using theelectropipettor, which can be operated to expel a small volume of fluidonto the dried reagent, followed by pipetting the expelled, fluidcomprising the reagent into the electropipettor. See also, U.S. Ser. No.08/671,986.

[0122] Generally, samples are optionally applied to the sample matrix byany of a number of well known methods. For example, sample libraries arespotted on sheets of a sample matrix using robotic pipetting systemswhich allow for spotting of large numbers of compounds. Alternatively,the sample matrix is treated to provide predefined areas for samplelocalization, e.g., indented wells, or hydrophilic regions surrounded byhydrophobic barriers, or hydrophobic regions surrounded by hydrophilicbarriers (e.g., where samples are originally in a hydrophobic solution),where spotted materials will be retained during the drying process. Suchtreatments then allow the use of more advanced sample applicationmethods, such as those described in U.S. Pat. No. 5,474,796, wherein apiezoelectric pump and nozzle system is used to direct liquid samples toa surface. Generally, however, the methods described in the '796 patentare concerned with the application of liquid samples on a surface forsubsequent reaction with additional liquid samples. However, thesemethods are readily modified to provide dry spotted samples on asubstrate. Similarly, the use of ink-jet printing technology to printbiological and chemical reagents onto substrates is well developed. See,e.g., Wallace (1996) Laboratory Automation News 1(5):6-9 where ink-jetbased fluid microdispensing for biochemical applications is described.

[0123] Similarly, cleavable linkers attaching compounds to an array canbe used to store the compounds in an array, followed by cleavage fromthe array. A variety of cleavable linkers, including acid cleavablelinkers, light or “photo” cleavable linkers and the like are known inthe art. Exemplar arrays are described in Pirrung et al., U.S. Pat. No.5,143,854 (see also, PCT Application No. WO 90/15070), Fodor et al., PCTPublication No. WO 92/10092 Fodor et al. (1991) Science, 251: 767-777;Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719; Kozal et al.(1996) Nature Medicine 2(7): 753-759 and Hubbell U.S. Pat. No.5,571,639. Immobilization of assay components in an array is typicallybe via a cleavable linker group, e.g., a photolabile, acid or baselabile linker group. Accordingly, the assay component is typicallyreleased from the assay e.g., by exposure to a releasing agent such aslight, acid, base or the like prior to flowing the test compound downthe reaction channel. Typically, linking groups are used to attachpolymers or other assay components during the synthesis of the arrays.Thus, preferred linkers operate well under organic and/or aqueousconditions, but cleave readily under specific cleavage conditions. Thelinker is optionally provided with a spacer having active cleavablesites. In the particular case of oligonucleotides, for example, thespacer is selected from a variety of molecules which can be used inorganic environments associated with synthesis as well as aqueousenvironments, e.g., associated with nucleic acid binding studies.Examples of suitable spacers are polyethyleneglycols, dicarboxylicacids, polyamines and alkylenes, substituted with, for example, methoxyand ethoxy groups. Linking groups which facilitate polymer synthesis onsolid supports and which provide other advantageous properties forbiological assays are known. In some embodiments, the linker providesfor a cleavable function by way of, for example, exposure to an acid orbase. Additionally, the linkers optionally have an active site on oneend opposite the attachment of the linker to a solid substrate in thearray. The active sites are optionally protected during polymersynthesis using protecting groups. Among a wide variety of protectinggroups which are useful are nitroveratryl (NVOC) α-methylnitroveratryl(Menvoc), allyloxycarbonyl (ALLOC), fluorenylmethoxycarbonyl (FMOC),α-methylnitro-piperonyloxycarbonyl (MeNPOC), —NH-FMOC groups, t-butylesters, t-butyl ethers, and the like. Various exemplary protectinggroups are described in, for example, Atherton et al., (1989) SolidPhase Peptide Synthesis, IRL Press, and Greene, et al. (1991) ProtectiveGroups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.

[0124] Other immobilization or spotting methods are similarly employed.For example, where samples are stable in liquid form, sample matricescan include a porous layer, gel or other polymer material which retain aliquid sample without allowing excess diffusion, evaporation or thelike, but permit withdrawal of at least a portion of the samplematerial, as desired. In order to draw a sample into an electropipettor,the pipettor will free a portion of the sample from the matrix, e.g., bydissolving the matrix, ion exchange, dilution of the sample, and thelike.

[0125] Whether the storage substrate is a filter, membrane, microtiterplate or other material holding reagents of interest, the substrate canconveniently be moved using a mechanical armature. Typically, thespatial location (or “physical address”) of the reagents on thesubstrate are known. The armature moves the substrate relative to themicrofluidic substrate (and electropipettor, where applicable) so thatthe component for transferring reagent from the substrate to thechannels and wells of a microfluidic substrate (e.g., anelectropipettor) contacts the desired reagent. Alternatively, themicrofluidic substrate or electropipettor can be moved by an armaturerelative to the storage substrate to achieve the same effect. Similarly,both the storage substrate and the microfluidic substrate can be movedby the mechanical armature to achieve the same effect. In anotheraspect, the microfluidic substrate, storage substrate or transferringcomponent (e.g., electropipettor) can be manually manipulated by theoperator.

[0126] A variety of electropipettors, including “resolubilization”pipettors for solubilizing dried reagents for introduction intomicrofluidic apparatus are described in Ser. No. 08/671,986, supra. Inbrief, an electropipettor pipettor having separate channels is fluidlyconnected to an assay portion of the microfluidic device (i.e., amicrofluidic substrate having the reaction and/or analysis and/orseparation channels, wells or the like). In one typical embodiment, theelectropipettor has a tip fluidly connected to a channel underelectroosmotic control. The tip optionally includes features to assistin sample transfer, such as a recessed region to aid in dissolvingsamples. Fluid can be forced into or out of the channel, and thus thetip, depending on the application of current to the channel. Generally,electropipettors utilize electrokinetic or “electroosmotic” materialtransport as described herein, to alternately sample a number of testcompounds, or “subject materials,” and spacer compounds. The pipettorthen typically delivers individual, physically isolated sample or testcompound volumes in subject material regions, in series, into the samplechannel for subsequent manipulation within the device. Individualsamples are typically separated by a spacer region of low ionic strengthspacer fluid. These low ionic strength spacer regions have highervoltage drop over their length than do the higher ionic strength subjectmaterial or test compound regions, thereby driving the electrokineticpumping, and preventing electrophoretic bias. On either side of the testcompound or subject material region, which is typically in higher ionicstrength solution, are fluid regions referred to as first spacer regions(also referred to as high salt regions or “guard bands”), that contactthe interface of the subject material regions. These first spacerregions typically comprise a high ionic strength solution to preventmigration of the sample elements into the lower ionic strength fluidregions, or second spacer region, which would result in electrophoreticbias. The use of such first and second spacer regions is described ingreater detail in U.S. patent application Ser. No. 08/671,986, supra.Spacers are not, however, required, particularly in those embodimentswhere transported components such as primers have the same charge andmass. It will be appreciated that embodiments using identically (ornearly identically) sized primers, such as modular primers, can be usedwithout guard bands.

[0127] In an additional aspect, the present invention provides kitsembodying the methods and apparatus herein. Kits of the inventionoptionally comprise one or more of the following: (1) an apparatus orapparatus component as described herein; (2) instructions for practicingthe methods described herein, and/or for operating the apparatus orapparatus components herein; (3) one or more assay component; (4) acontainer for holding apparatus or assay components, and, (5) packagingmaterials.

[0128] In a further aspect, the present invention provides for the useof any apparatus, apparatus component or kit herein, for the practice ofany method or assay herein, and/or for the use of any apparatus or kitto practice any assay or method herein.

EXAMPLES

[0129] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill will readily recognize avariety of noncritical parameters, which are changed or modified toyield essentially similar results.

Example 1 Integrated Analysis on Multi-phasic Microfluidic Apparatus

[0130] This example describes three experiments in which multipleoperations in a biochemical assay were run on a chip. It demonstratesthe ability to integrate functions such as complex (blood) samplepreparation, specialized reactions (polymerase chain reaction, PCR), andsophisticated analysis (DNA size separation) in a single format on amulti-phasic microfluidic apparatus.

[0131] In the first experiment we used a Caliper LabChip™ to load DNAtemplate, run the PCR reaction, and then size the resulting PCR productby gel separation. In the second experiment we used whole blood as thesource of DNA. The third experiment also starts with whole blood with analternate method of sample preparation.

[0132] All three experiments were done on a “12A” chip which is shown onthe right-hand-side of FIG. 3. The entire chip was first filled with asieving matrix gel. The cross-channel fluid was replaced with PCR mixsuitable to amplify an approximately 500 bp segment of the bacteriophageLambda. The loaded chip was placed on a thermocycler (MJ Research) andthe temperature cycled to amplify the DNA in the wells and the channel.At the end of the cycling procedure, the chip was placed on a microscopedetection station and the product was electrokinetically injected intothe sieving channel. The peak which is shown in the accompanyingelectropherogram on FIG. 3 at 27-28 seconds corresponds to the Lambda500-bp product.

[0133] In the second experiment whole blood mixed with PCR reaction mix(1% blood) was placed in the wells of the same type of chip. The chipwas placed on a thermal cycler and cycled with a few additional steps atthe beginning to aid in the amplification of blood. The amplifiedproduct was separated in the gel-filled channel and the product of thereaction is shown in FIG. 4. A negative control in which no blood wasadded to the mix was run with the same procedure and no peak is seen inthat run. The electropherograms showing the positive an negative runsare shown in FIG. 4.

[0134] The third experiment also used the 12A chip but with all channelsfilled and remaining filled with sieving gel. In addition, the two wellsat the ends of the separation channel were filled with gel. For thefirst part of the experiment, approximately 2000 lymphocytes (whiteblood cells) purified from whole blood in a conventional way(centrifugation) were added to 20 μL of PCR reaction mix and placed inthe sample well of the chip. The wells were overlaid with mineral oiland the chip was cycled using a thermocycler. After cycling, the PCRproduct was separated on a different but still cycled 12A chip. FIG. 5shows the electropherogram for this portion where the amplified peak ofthe HLA locus (about 300 bp) is seen at around 34 seconds at the sametime as the 270-310 bp fragments in the PhiX 174 standard ladder. Forthe second part of the experiment, PCR reaction mix without DNA templatewas placed in the sample well of a fresh chip and 5% whole blood inwhich the red blood cells had been lysed was placed in another well.Lymphocytes (white blood cells) were electrophoresed through the channelto the well containing the PCR reaction mixture until 20-100 lymphocyteswere in the PCR well. The chip was cycled and DNA separated as for theprevious chip. This is shown in FIG. 6. Amplification was achieved forboth purified and electrophoresed lymphocytes, although the amount ofproduct for purified lymphocytes was larger than for electrophoresedlymphocytes as shown in FIG. 7 in which the two product peaks areoverlaid. Sufficient PCR cycles were run to ensure that the reaction hadreached a plateau stage, because the number of starting copies wasdifferent.

[0135] These experiments demonstrated the ability to integrate severalsteps of a complex biochemical assay in a multiphasic microchip format.

Example 2 PCR Compatibility with Separations Gel

[0136] A simplified chip for PCR/DNA separation using joule heating forPCR includes a continuous fluid phase throughout the chip such that PCRamplification is carried out in one section of the chip by joule heatingand DNA separation is performed in another. This example shows that thepolymerase chain reaction (PCR) can be run in the presence of a DNAseparations gel in a microfluidic system, and that the resulting nucleicacid DNA products are separable in the gel in a microfluidic system.

[0137] In this example, the 12 A chip described above was filled withone of 5 media comprising PCR reaction reagents. The 5 media which weretested are: 1.7% Agarose; 5% linear polyacrylamide (PA); 0.5% Methylcellulose; 2.5% polyethylene oxide (PEO), and 0.5% hydroxy cellulose(HEC). Solutions containing the separation media were prepared in diH₂Osuch that the final concentration in the PCR mixture was the appropriateconcentration for DNA separation: Sieving medium Final Concentrationg/mL DNA agarose 1.7% 0.45 Linear PA   5% .131 Methyl cellulose 0.5%.131 PEO 2.5% .065 HEC 0.5% .013

[0138] The PCR master mix was prepared as follows: H₂O + sieving matrix36.3 μL   buffer 10 μL dNTPs 10 μL triton 10 μL BSA 10 μL MgCl₂ 10 μLHLA P1  3 μL HLA P2  3 μL 1/10 YP  1 μL UNG  1 μL Taq gold 0.7 μL 

[0139] The positive control for PCR was 47.5 μL of the above master mixwith 2.5 μL 1× DNA. The negative control was 47.5 μL of the above mastermix with 2.5 μL H₂O (both cycled in standard PCR tubes). PCR reactionconditions were at 94° C. (15 minutes); 94° C. (30 seconds); 55° C. (30seconds); 65° C. (60 seconds). Results showed that PCR amplification wassuccessful in the presence of each of the sieving matrices describedabove (PEO and HEC were slightly inhibitory).

[0140] Once PCR in the presence of the various sieving matrixes wasshown to be possible, separation of the PCR fragments in the sievingmatrix in 1× PCR buffer was investigated on the 12 A chip. The PCRmixture was amplified in the presence of 0.5% MC. The 0.5% MC sievingmatrix was dispersed throughout the 12 A chip discussed above. PCRproducts and phi X control DNA were separated in the channels of the 12A chip, by applying current to wells as described below: Script wellnumber 1 2 7 8 time −1.3 μA 2000 v −1.3 μA −6 μA  45 sec 1000 v 4 μA2000 v 4 μA  1 sec 500 v 0.5 μA 10 μA 0.5 μA 180 sec

[0141] The phi X control was 2.5 μL phi-X DNA (i.e., Phi-X 174 digestedto completion with Hae III, available from Promega) with 1 μL 1/100sybergreen and 10 μL 10× PCR buffer, plus 86.5 μL H₂O. FIG. 8 shows theepifluorescent profile of the PCR product and phi X control. Intensityunits are arbitrary. As shown, PCR products made in the presence of 0.5%methyl cellulose were resolved in channels as described.

[0142] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques and apparatusdescribed above may be used in various combinations. All publicationsand patent documents cited in this application are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent document were so individuallydenoted.

What is claimed is:
 1. A microfluidic apparatus comprising: amicrofluidic substrate comprising a first channel intersecting a secondchannel, wherein the first channel contains a first fluidic phase andthe second channel contains a second fluidic phase, wherein the firstand second fluidic phases cross and are substantially unmixed at theintersection between the channels.
 2. The microfluidic apparatus ofclaim 1, wherein the intersection between the first and second channelis unvalved or electrically gated.
 3. The microfluidic apparatus ofclaim 1, wherein the first fluidic phase provides for electoosmoticmovement of a component in the first phase and the second fluidic phaseprovides for electrophoretic movement of the component in the secondphase.
 4. The microfluidic apparatus of claim 1, wherein the first orsecond fluidic phase is electrically loaded in the first or secondchannel.
 5. A microfluidic apparatus comprising: a microfluidicsubstrate comprising a first channel intersecting a second channel,wherein the first channel comprises an electroosmotic fluidic phase andthe second channel comprises an electrophoretic fluidic phase and theintersection between the first and second channel is unvalved orelectrically gated.
 6. The microfluidic apparatus of claim 5, whereinthe electrophoretic fluid phase comprises a sieving matrix.
 7. Themicrofluidic apparatus of claim 1 or 5, the apparatus further comprisinga multiport electrical controller which is in fluid communication withthe first and second channels.
 8. The microfluidic apparatus of claim 7,wherein the electrical controller provides electroosmotic control of acomponent in the first channel and electrophoretic control of thecomponent in the second channel.
 9. The microfluidic apparatus of claim7, wherein the electrical controller provides crossed electroosmotic andelectrophoretic control of one or more components in the first andsecond channels.
 10. The microfluidic apparatus of claim 5, wherein theintersection of the first and second channels is substantially free ofthe electrophoretic phase.
 11. The microfluidic apparatus of claim 10,wherein molecular components stack against the electrophoretic phase inthe second channel at an edge of the intersection between the first andsecond channels.
 12. The microfluidic apparatus of claim 1 or 5, whereinan interior portion of the first or second channel has at least onecross sectional dimension between about 0.1 μm and 500 μm.
 13. Themicrofluidic apparatus of claim 5, wherein the electrophoretic phase orthe electroosmotic phase are electrically loaded in the first or secondchannel.
 14. The microfluidic apparatus of claim 1 or 5, furthercomprising a third channel in fluid communication with the first orsecond channel.
 15. The microfluidic apparatus of claim 1 or 5, furthercomprising a high resistance channel region for joule heating.
 16. Themicrofluidic apparatus of claim 1 or 5, wherein the first channelcomprises a resistance region for joule heating and wherein theapparatus comprises PCR reagents in the first channel.
 17. Themicrofluidic apparatus of claim 1 or 5, wherein the microfluidicsubstrate comprises a pressure port in fluid communication with thefirst or second channel.
 18. The microfluidic apparatus of claim 1 or 5,further comprising a microfluidic substrate holder for mounting thesubstrate.
 19. The microfluidic apparatus of claim 1 or 5, furthercomprising a component detection region in a channel wall and a detectormounted to view a component.
 20. The microfluidic apparatus of claim 1or 5, further comprising a component detection region in a channel wall,a detector mounted to view a component, a recording device operablylinked to the detector, and a digital computer operably linked to therecording device.
 21. A microfluidic apparatus comprising at least twointersecting channels in fluid communication, wherein the twointersecting channels are substantially filled with an electrophoreticphase and wherein the electrophoretic phase comprises at least tworeactants dispersed in the electrophoretic phase.
 22. The microfluidicapparatus of claim 21, wherein the electrophoretic phase is a sievingmatrix.
 23. The microfluidic apparatus of claim 21, wherein theapparatus comprises an electrical controller which directselectrophoretic movement of the at least two reactants in theelectrophoretic phase.
 24. The microfluidic apparatus of claim 21,wherein the apparatus comprises a reaction region.
 25. The microfluidicapparatus of claim 21, wherein the apparatus comprises a high-resistancechannel region for heating the at least two reactants.
 26. Themicrofluidic apparatus of claim 21, wherein the at least two reactantsare heterogeneously dispersed throughout at least a portion of theelectrophoretic phase.
 27. The microfluidic apparatus of claim 21,wherein the at least two reactants are homogeneously dispersedthroughout the electrophoretic phase.
 28. The microfluidic apparatus ofclaim 21, wherein one of the at least two reactants is homogeneouslydispersed throughout the electrophoretic phase and one of the reactantsis heterogeneously dispersed in at least a portion of theelectrophoretic phase.
 29. The microfluidic apparatus of claim 21,wherein the channels comprise a region with an interior dimension ofbetween about 0.1 μm and 500 μm.
 30. The microfluidic apparatus of claim21, wherein the reactants comprise PCR reagents.
 31. A method of makinga microfluidic substrate comprising: fabricating at least a first andsecond channel in the microfluidic substrate, wherein an interiorportion of the first or second channel has at least one cross sectionaldimension between about 0.1 μm and 500 μm and wherein the first andsecond channels intersect; and, selectively fixing an electrophoreticphase in a selected region of the first or second channel.
 32. Themethod of claim 31, wherein the intersection of the first and secondchannels is unvalved or electrically gated.
 33. The method of claim 31,wherein the electrophoretic phase is electrically loaded.
 34. The methodof claim 31, wherein the electrophoretic phase is loaded under pressure.35. The method of claim 31, wherein the electrophoretic phase is asieving matrix.
 36. The method of claim 31, wherein the electrophoreticphase is selectively fixed by photopolymerizing the electrophoreticphase in the selected region.
 37. A method of making a microfluidicsubstrate comprising an electrophoretic phase in a selected region of amicrofluidic channel in the microfluidic substrate, the methodcomprising: providing a microfluidic substrate comprising at least onemicrofluidic channel intersection; flowing a component of theelectrophoretic phase into at least the selected region of at least oneof the intersecting channels; and fixing the component in the selectedregion, thereby providing the electrophoretic phase in the selectedregion.
 38. The method of claim 37 wherein the electrophoretic phase isa sieving matrix.
 39. The method of claim 31 or claim 37, wherein theelectrophoretic phase is fixed by replacing a portion of theelectrophoretic phase with a selected fluid.
 40. The method of claim 39,wherein replacement of the electrophoretic phase with the selected fluidis performed by introducing the selected fluid into a region of thefirst or second channel under pressure.
 41. The method of claim 31 or37, wherein the electrophoretic phase is photopolymerizable, and fixingthe component in the selected region comprises exposing the selectedregion to light, thereby polymerizing the component to provide theelectrophoretic phase in the selected region.
 42. The method of claim 31or 37, the method comprising flowing a cross-linking or activatingcompound into the selected region whereby the cross-linking oractivating compound results in polymerization of the component toprovide the electrophoretic phase in the selected region.
 43. The methodof claim 42, wherein the electrophoretic phase is polyacrylamide and theactivating or cross linking compound comprises TEMED.
 44. The method ofclaim 31 or 37, wherein the component of the electrophoretic phase isflowed into multiple regions of one or more channels in the microfluidicsubstrate including the selected region and is selectively fixed inplace in the selected region.
 45. The method of claim 44, the methodcomprising flowing a solubilization compound into an unselected regionof a channel, thereby dissolving the electrophoretic phase in theunselected region.
 46. The method of claim 45, wherein thesolubilization compound comprises sodium periodate.
 47. The method ofclaim 37, the method comprising dispersing reactant components in thecomponent of the electrophoretic phase and electrophoresing the reactantcomponents into the selected region, whereby the reactant components areconcentrated in the selected region to provide concentrated reactioncomponents.
 48. The method of claim 47, wherein the concentratedreaction components are reacted.
 49. The method of claim 48, wherein thereaction components are reacted by heating the components.
 50. Themethod of claim 48, wherein the reaction components comprise PCRreagents and the method comprises cycled heating of the components. 51.A microfluidic apparatus for performing PCR comprising a firstmicrochannel having disposed therein a mixture comprising a sievingmatrix and a plurality of PCR reaction components.
 52. The apparatus ofclaim 51, further comprising means for heating the mixture in thechannel.
 53. The apparatus of claim 51, further comprising a secondmicrochannel intersecting the first microchannel, wherein the firstmicrochannel comprises a resistance region for joule heating and thesecond channel comprises a detection region for detecting a PCR product.54. The apparatus of claim 53, the apparatus further comprising afluorescence detector positioned to view the detection region.
 55. Theapparatus of claim 51, the wherein the plurality of PCR reactioncomponents comprises a plurality of: a thermostable DNA polymerase, aplurality of nucleotides, a nucleic acid template, and at least oneprimer which hybridizes to the nucleic acid template.
 56. The apparatusof claim 55, wherein the plurality of PCR reaction components comprisesa thermostable DNA polymerase, a plurality of nucleotides, a nucleicacid template, at least one primer which hybridizes to the nucleic acidtemplate and Mg++.
 57. The apparatus of claim 55, wherein the nucleicacid template is a DNA template.
 58. The apparatus of claim 55, theapparatus further comprising an electrokinetic direction system forelectrophoretic movement of charged molecules in the microchannel. 59.The apparatus of claim 58, wherein the electrokinetic direction systemis used for joule heating of the PCR reaction mixture.
 60. The apparatusof claim 51, wherein the sieving matrix is selected from the groupconsisting of agarose, linear polyacrylamide, methylcellulose,polyethylene oxide and hydroxy ethyl cellulose.
 61. A method ofperforming PCR comprising: mixing components of a PCR reaction mixturewith a sieving matrix to provide a PCR sieving matrix mixture; and,thermocycling the resulting PCR sieving matrix mixture to produce a PCRproduct.
 62. The method of claim 61, wherein the components of the PCRreaction mixture are mixed with the sieving matrix in a microfluidicchannel.
 63. The method of claim 62, further comprising joulethermocycling of the PCR reaction mixture in the channel to produce thePCR product.
 64. The method of claim 62, further comprisingelectrophoresing the PCR product in the microfluidic channel.
 65. Themethod of claim 62, wherein the PCR product is electrokineticallytransported into a second microfluidic channel which intersects thefirst microfluidic channel.
 66. The method of claim 61, wherein thesieving matrix is selected from the group consisting of agarose, linearpolyacrylamide, methylcellulose, polyethylene oxide and hydroxy ethylcellulose.